Method for quantitatively detecting antigen

ABSTRACT

A method for quantitatively detecting an antigen which comprises (1) a first step of providing an Fab′ antibody having a uniform isoelectric point, said antibody forming an immune complex with an antigen in an analytical sample and being modified by adding an amino acid sequence comprising a charged amino acid residue and by being labeled with a fluorescent dye, (2) a second step of mixing the Fab′ antibody having a uniform isoelectric point with the analytical sample containing the antigen to obtain a mixture comprising the immune complex, (3) a third step of separating the mixture by performing electrophoresis in a carrier, (4) a fourth step of irradiating an excitation light which excites the fluorescent dye to the mixture separated in the third step to cause fluorescence in the immune complex, and (5) a fifth step of detecting the fluorescence.

TECHNICAL FIELD

This invention relates to a method for quantitatively detecting anantigen, more specifically, to a method for quantitatively detecting anantigen using an Fab′ antibody having a uniform isoelectric point, saidantibody forming an immune complex with an antigen in an analyticalsample and being modified by adding an amino acid sequence comprising acharged amino acid residue and by being labeled with a fluorescent dye.

BACKGROUND ART

When electric field is supplied to a charged substance in an electrolytesolution, the substance migrates toward the electrode having an oppositecharge of the substance. This phenomenon, electrophoresis, is widelyused as a means for separating various substances. Generally,electrophoresis of an analytical sample is performed in a carrier havinga constant pH. On the contrary, a carrier having a gradient pH is usedwhen electrophoresis is performed based on the isoelectric focusingmethod. Since the isoelectric focusing method was developed, it has beenacquiring popularity as a means for separating amphoteric electrolytes,such as amino acids and proteins.

An amphoteric electrolyte has a pH value where its effective chargebecomes zero, and the pH value is called an isoelectric point. Whenelectrophoresis of the analytical sample consisting of an amphotericelectrolyte is performed based on the isoelectric focusing method, thesample stops at a certain position in an electrophoresis carrier and isconcentrated there. This position is where a pH value of theelectrophoresis carrier is equal to the isoelectric point of the sample.In this case, the separated sample is concentrated in a focusing manner,therefore, the isoelectric focusing method has very high separability.

Recently, the capillary electrophoresis, where electrophoresis isperformed in a capillary having an inside diameter of several 10micrometers and a length of several 100 milimeters, was established.Using this method, very high separability is obtained with a very smallamount of analytical sample. Therefore, the method is applied toseparation and analysis of various samples including proteins, inorganicions, low molecular compounds, nucleic acids, and the like. When thecapillary electrophoresis is performed, concentration of the separatedanalytical sample can be quantified using a detector equipped at one endof the capillary.

Detection of the sample separated by the electrophoresis is generallydone by the ultraviolet/visible detecting method that includesirradiation of an ultraviolet light or a visible light to the sample andmeasurement of the changes of the amount of light absorbed. Detectioncan be done with higher sensitivity when a fluorescence detection methodis applied. In the fluorescence detection method, an analytical sampleis labeled with a fluorescent dye (fluorescently labeling). In thismethod, an excitation light is focused on the separated sample, andfluorescence generated is detected to quantify the concentration of thesample.

It is possible to combine above-mentioned isoelectric focusing method,capillary electrophoresis, and fluorescence detection method. Thecombined method is called fluorescently detecting capillary isoelectricfocusing. In the case of fluorescently detecting capillary isoelectricfocusing, electrophoresis is performed to the fluorescently labeledsample in a carrier having a gradient pH, which is held in thecapillary, and the fluorescence caused by irradiation of an excitationlight is detected with an optical detector or the like. According tothis method, even with the sample of a very small amount, a highlyprecise quantitative detection is possible. Therefore, this methodattracts attention as a super-high-sensitive analytical method forproteins or the like.

In recent years, in many cases, a minor constituent in a living body isanalyzed by the electrophoresis mentioned above. When performing suchanalysis, an immune complex formed by reacting a minor constituent in aliving body with the antibody that recognizes the minor constituent asan antigen is detected. The immune complex is preferably labeled with afluorescent dye for the purpose of accurate detection. In this case,either the antigen or the antibody needs to be fluorescently labeled.Upon labeling the antigen or the antibody using the fluorescent dye,conventional labeling method can not be applied for the followingreasons.

Antibodies and many antigens are composed of proteins. The number ofamino groups at an N-terminal of a protein and at a lysine side chain,and the dissociated state thereof are great factors in determining theisoelectric point of a protein (Zokuseikagaku Jikkenkoza 2, Chemistry ofproteins, volume 1, Society of Japan Biochemistry, 1987). Therefore, theconventional labeling method utilizing a reaction between a fluorescentdye and an amino group of a protein greatly changes the isoelectricpoint of a protein.

In addition, the number and position of a fluorescent dye bound to aprotein become indefinite because there are a large number of aminoacids that are reactive with a fluorescent dye in a protein.Accordingly, the resultant protein becomes a mixture of proteins showingdifferent isoelectric point, which makes it difficult to conduct aprecise analysis using the isoelectric focusing. Further, since thethree-dimensional structure of a protein is changed by a fluorescentdye, there is also a problem that the chemical stability of a proteinitself is deteriorated.

In addition, there is a situation where an analysis by isoelectricfocusing can not be done with high accuracy even when a monoclonalantibody having a uniform molecular weight obtained by a hybridoma isused as an antibody for detection. This is because the monoclonalantibody having a uniform molecular weight produced by hybridoma doesnot necessarily have a uniform isoelectric point, and this phenomenon iscalled microheterogeneity (Bouman H et al., Z Immunitatsforsch Exp KlinImmunol. 1975 October; 150 (4): 370–7).

As reasons for this non-uniformity of isoelectric point, deamidation ofa protein (Robinson A. B. et al., Proc. Natl. Acad. Sci. U.S.A. 1970July; 66 (3): 753–7), pyroglutamylation of an N-terminal (Scott D. I. Etal., Biochem J. 1972 August; 128(5): 1221–7), addition of a sugar chain(Cohenford M. A. et al. Immunol. Commun. 1983; 12(2): 189–200),myristoylation (Pillai S. et al., Proc. Natl. Acad. Sci. U.S.A. 1987November; 84(21): 7654–8) and the like have been proposed, but themechanism for the non-uniformity of the isoelectric point of a proteinhas not been specified yet.

Therefore, when an analytical sample shows plural isoelectric points byisoelectric focusing, it is difficult to determine where the pluralityoriginates from. This is because the plurality is ascribable either toan antigen or to an antibody. Both antigen and antibody may havenon-uniformity of isoelectric point, as mentioned above.

Therefore, when analysis is performed by electrophoresis utilizing anantigen-antibody reaction, an antibody is necessary to be uniform interms of isoelectric point, and when an antibody having a uniformisoelectric point is fluorescently labeled with a fluorescent dye, thefluorescent dye should not be reacted with an amino group of theantibody as described above.

Shimura K. and Karger B. L. disclose a method for quantitativelydetecting an antigen using an antibody having a uniform isoelectricpoint, (see Anal. Chem. 1994 Jan. 1; 66(1): 9–15, or JP-A 8-506182). Themethod disclosed in these references is schematically shown in FIGS. 8Ato G. That is, IgG antibody produced by a hybridoma (FIG. 8A) is cutwith a protease (pepsin) and the resulting F (ab′)₂ antibody (FIG. 8B)is separated. This is treated with a reducing agent ofmercaptoethylamine to reduce three disulfide bonds (S—S bond) to obtainFab′ antibody (FIG. 8C). This Fab′ antibody is oxidized to leave behindonly one reactive thiol group (SH group) (FIG. 8D) and a fluorescent dyeis bound to this thiol group (FIG. 8E). The resulting fluourescentlylabeled Fab′ antibody is separated by isoelectric focusing and afluorescently labeled Fab′ antibody having a uniform isoelectric pointis taken from an electrophoresis carrier (FIG. 8F). The obtainedfluorescently labeled Fab′ antibody having a uniform isoelectric pointis combined with an antigen. Then, electrophoresis is performed, andfluorescence caused by excitation light is measured. (FIG. 8G).

DISCLOSURE OF THE INVENTION

However, the method disclosed by Shimura K and Karger B L, as above,involve steps for obtaining Fab′ antibody having a uniform isoelectricpoint that are complicated. In addition, when an isoelectric point ofthe antigen, which is the analyte, is close to an isoelectric point ofthe fluorescently labeled antibody, migration time of the immune complexcomprising antigen and antibody becomes almost the same as that ofexcessive antigen and/or antibody. Therefore, the electrophoretic peaksoverlap and detection can not be performed with high accuracy.

The present invention was done in view of the aforementioned problems ofthe prior art and an object of the present invention is to provide amethod for quantitatively detecting an antigen which enables theanalysis of the antigen with high accuracy, even when the isoelectricpoint of the antigen as the analyte is close to that of thefluorescently labeled antibody.

The present inventors studied extensively and, as a result, found thatit is possible to analyze an antigen with high accuracy by using an Fab′antibody having a uniform isoelectric point, which has been modified byadding an amino acid sequence comprising a charged amino acid residueand by being labeled with a fluorescent dye, and which forms an immunecomplex with an antigen in an analytical sample.

That is, the present invention provides a method for quantitativelydetecting an antigen which comprises (1) a first step of providing anFab′ antibody having a uniform isoelectric point, said antibody formingan immune complex with an antigen in an analytical sample and beingmodified by adding an amino acid sequence comprising a charged aminoacid residue and by being labeled with a fluorescent dye, (2) a secondstep of mixing the Fab′ antibody having a uniform isoelectric point withthe analytical sample containing the antigen to obtain a mixturecomprising the immune complex, (3) a third step of separating themixture by performing electrophoresis in a carrier, (4) a fourth step ofirradiating an excitation light which excites the fluorescent dye to themixture separated in the third step to cause fluorescence in the immunecomplex, and (5) a fifth step of detecting the fluorescence.

In a method for quantitatively detecting an antigen of the presentinvention, it is preferred that the amino acid sequence is addedadjacent to a C-terminal of an L chain of the Fab′ antibody having auniform isoelectric point. And it is preferred that the fluorescent dyeis bound to a cysteine residue which is not involved in binding with anL chain and which exists in an amino acid sequence adjacent to aC-terminal of a CH1 region of the Fab′ antibody having a uniformisoelectric point.

In addition, in a method for quantitatively detecting an antigen of thepresent invention, it is preferred that the electrophoresis is performedby isoelectric focusing and that the electrophoresis is performed bycapillary electrophoresis.

Further, it is preferred that the Fab′ antibody having a uniformisoelectric point is produced by a method which comprises (1) a firststep of providing an Fd chain gene encoding a VH region, a CH1 region,and an amino acid sequence which adjoins to a C-terminal of the CH1region and comprises a cysteine residue which is not involved in bindingwith an L chain in an Fab′ antibody, (2) a second step ofsite-specifically mutating in the Fd chain gene at least one codonencoding an amide group-containing amino acid residue in the CH1 region,into a codon encoding an amide group-non-containing amino acid residueexcept for cysteine to obtain a modified Fd chain gene, (3) a third stepof linking the modified Fd chain gene and an L chain gene encoding an Lchain of the Fab′ antibody in the expressible state to obtain a geneexpressing a modified Fab′ antibody, (4) a fourth step of modifying thegene expressing a modified Fab′ antibody to express an amino acidsequence comprising a charged amino acid residue adjacent to aC-terminal of the L chain to obtain a gene expressing, a charge modifiedFab′ antibody, (5) a fifth step of transforming a host cell with thegene expressing a charge modified Fab′ antibody and culturing theresultant transformant to obtain an Fab′ antibody having a uniformisoelectric point, the Fab′ antibody being modified by adding an aminoacid sequence comprising a charged amino acid residue adjacent to theC-terminal of the L chain, and by adding an amino acid sequencecomprising a cysteine residue which is not involved in binding with an Lchain adjacent to the C-terminal of the CH1 region, and (6) a sixth stepof binding a fluorescent dye to the cysteine residue which is notinvolved in binding with an L chain in the Fab′ antibody having auniform isoelectric point obtained in the fifth step.

In addition, it is preferred that the Fab′ antibody having a uniformisoelectric point is produced by a method which comprises (1) a firststep of providing an Fd chain gene encoding a VH region, a CH1 region,and an amino acid sequence which adjoins to a C-terminal of the CH1region and comprises a cysteine residue which is not involved in bindingwith an L chain in an Fab′ antibody, (2) a second step ofsite-specifically mutating in the Fd chain gene at least one codonencoding an amide group-containing amino acid residue in the CH1 region,into a codon encoding an amide group-non-containing amino acid residueexcept for cysteine to obtain a modified Fd chain gene, (3) a third stepof providing an L chain gene encoding an L chain of the Fab′ antibody,(4) a fourth step of modifying the L chain gene to express an amino acidsequence comprising a charged amino acid residue adjacent to aC-terminal of the L chain to obtain a charge modified L chain gene, (5)a fifth step of linking the modified Fd chain gene and the chargemodified L chain gene in the expressible state to obtain a geneexpressing a charge modified Fab′ antibody, (6) a sixth step oftransforming a host cell with the gene expressing a charge modified Fab′antibody and culturing the resultant transformant to obtain an Fab′antibody having a uniform isoelectric point, the Fab′ antibody beingmodified by adding an amino acid sequence comprising a charged aminoacid residue adjacent to the C-terminal of the L chain, and by adding anamino acid sequence comprising a cysteine residue which is not involvedin binding with an L chain adjacent to the C-terminal of the CH1 region,and (7) a seventh step of binding a fluorescent dye to the cysteineresidue which is not involved in binding with an L chain in the Fab′antibody having a uniform isoelectric point obtained in the sixth step.

Further, it is preferred that the Fab′ antibody having a uniformisoelectric point is produced by a method which comprises (1) a firststep of providing an Fd chain gene encoding a VH region, a CH1 region,and an amino acid sequence which adjoins to a C-terminal of the CH1region and comprises a cysteine residue which is not involved in bindingwith an L chain in an Fab′ antibody, and an L chain gene encoding the Lchain of the Fab′ antibody, (2) a second step of linking the Fd chaingene and the L chain gene in the expressible state to obtain a geneexpressing an Fab′ antibody, (3) a third step of modifying the geneexpressing an Fab′ antibody to express an amino acid sequence comprisinga charged amino acid residue adjacent to a C-terminal of the L chain,and site-specifically mutating in the gene expressing an Fab′ antibodyat least one codon encoding an amide group-containing amino acid residuein the CH1 region, into a codon encoding an amide group-non-containingamino acid residue except for cysteine to obtain a gene expressing acharge modified Fab′ antibody, (4) a fourth step of transforming a hostcell with the gene expressing a charge modified Fab′ antibody andculturing the resultant transformant to obtain an Fab′ antibody having auniform isoelectric point, the Fab′ antibody being modified by adding anamino acid sequence comprising a charged amino acid residue adjacent tothe C-terminal of the L chain, and by adding an amino acid sequencecomprising a cysteine residue which is not involved in binding with an Lchain adjacent to the C-terminal of the CH1 region, and (5) a fifth stepof binding a fluorescent dye to the cysteine residue which is notinvolved in binding with an L chain in the Fab′ antibody having auniform isoelectric point obtained in the fourth step.

Furthermore, it is preferred that the Fab′ antibody having a uniformisoelectric point is produced by a method which comprises (1) a firststep of providing a CH1 gene encoding a CH1 region and an amino acidsequence which adjoins to a C-terminal of the CH1 region and comprises acysteine residue which is not involved in binding with an L chain in afirst Fab′ antibody, and a CL gene encoding a CL region of the firstFab′ antibody, (2) a second step of site-specifically mutating in theCH1 gene at least one codon encoding an amide group-containing aminoacid residue in the CH1 region, into a codon encoding an amidegroup-non-containing amino acid residue except for cysteine to obtain amodified CH1 gene, (3) a third step of cutting the modified CH1 genewith a restriction enzyme to obtain a gene fragment encoding the CH1region, (4) a fourth step of providing a VH gene encoding a VH region ofa second Fab′ antibody and a VL gene encoding a VL region of the secondFab′ antibody, (5) a fifth step of linking the gene fragment, the CLgene, the VH gene and the VL gene in the expressible state to obtain agene expressing a modified Fab′ antibody, (6) a sixth step of modifyingthe gene expressing a modified Fab′ antibody to express an amino acidsequence comprising a charged amino acid residue adjacent to aC-terminal of the CL region to obtain a gene expressing a chargemodified Fab′ antibody, (7) a seventh step of transforming a host cellwith the gene expressing a charge modified Fab′ antibody and culturingthe resultant transformant to obtain an Fab′ antibody having a uniformisoelectric point, the Fab′ antibody being modified by adding an aminoacid sequence comprising a charged amino acid residue adjacent to theC-terminal of the L chain, and by adding an amino acid sequencecomprising a cysteine residue which is not involved in binding with an Lchain adjacent to the C-terminal of the CH1 region, and (8) a eighthstep of binding a fluorescent dye to the cysteine residue which is notinvolved in binding with an L chain in the Fab′ antibody having auniform isoelectric point obtained in the seventh step.

In addition, it is preferred that the Fab′ antibody having a uniformisoelectric point is produced by a method which comprises (1) a firststep of providing a CH1 gene encoding a CH1 region and an amino acidsequence which adjoins to a C-terminal of the CH1 region and comprises acysteine residue which is not involved in binding with an L chain in afirst Fab′ antibody, and a CL gene encoding a CL region of the firstFab′ antibody, (2) a second step of site-specifically mutating in theCH1 gene at least one codon encoding an amide group-containing aminoacid residue in the CH1 region, into a codon encoding an amidegroup-non-containing amino acid residue except for cysteine to obtain amodified CH1 gene, (3) a third step of cutting the modified CH1 genewith a restriction enzyme to obtain a gene fragment encoding the CH1region, (4) a fourth step of modifying the CL gene to express an aminoacid sequence comprising a charged amino acid residue adjacent to aC-terminal of the CL region to obtain a charge modified CL gene, (5) afifth step of providing a VH gene encoding a VH region of a second Fab′antibody and a VL gene encoding a VL region of the second Fab′ antibody,(6) a sixth step of linking the gene fragment, the charge modified CLgene, the VH gene and the VL gene in the expressible state to obtain agene expressing a charge modified Fab′ antibody, (7) a seventh step oftransforming a host cell with the gene expressing a charge modified Fab′antibody and culturing the resultant transformant to obtain an Fab′antibody having a uniform isoelectric point, the Fab′ antibody beingmodified by adding an amino acid sequence comprising a charged aminoacid residue adjacent to the C-terminal of the L chain, and by adding anamino acid sequence comprising a cysteine residue which is not involvedin binding with an L chain adjacent to the C-terminal of the CH1 region,and (8) a eighth step of binding a fluorescent dye to the cysteineresidue which is not involved in binding with an L chain in the Fab′antibody having a uniform isoelectric point obtained in the seventhstep.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view schematically showing human IgG1 antibody.

FIG. 2A is a constituent view of the gene expressing the Fab′ antibodyhaving a uniform isoelectric point.

FIG. 2B is a view showing the pCANTAB5E plasmid vector to which the geneexpressing the Fab′ antibody having a uniform isoelectric point isintroduced.

FIG. 3 is a view showing migration time and fluorescent intensity whenfluorescently detecting capillary isoelectric focusing is performedusing the anti-human alpha-1-antitrypsin Fab′ antibody which is notmodified.

FIG. 4 is a view showing migration time and fluorescent intensity whenfluorescently detecting capillary isoelectric focusing is performedusing the modified anti-human alpha-1-antitrypsin Fab′ antibody (H-N162Dmodified Fab′ antibody).

FIG. 5 is a view showing migration time and fluorescence intensity whenelectrophoresis is performed to the immune complex comprising the Fab′antibody having a uniform isoelectric point and the antigen.

FIG. 6 is a view showing migration time and fluorescence intensity whenelectrophoresis is performed to the immune complex comprising the Fab′antibody having a uniform isoelectric point which is modified by addingan amino acid sequence comprising a charged amino acid residue, and theantigen.

FIG. 7A is a view schematically showing Escherichia coli in which thegene expressing the Fab′ antibody having a uniform isoelectric point isincorporated.

FIG. 7B is a view schematically showing the Fab′ antibody having auniform isoelectric point which was produced by antibody induction withIPTG.

FIG. 7C is a view schematically showing the fluorescently labeled Fab′antibody having a uniform isoelectric point.

FIG. 7D is a view schematically showing the relation of migration timeand fluorescence intensity obtained when electrophoresis is performed tothe immune complex comprising the fluorescently labeled Fab′ antibodyhaving a uniform isoelectric point and the antigen.

FIG. 8A is a view schematically showing IgG antibody produced by ahybridoma.

FIG. 8B is a view schematically showing the F(ab′)₂ antibody obtained bycutting IgG antibody produced by the hybridoma with a protease.

FIG. 8C is a view schematically showing the Fab′ antibody obtained byreducing disulfide bonds by treating F(ab′)₂ antibody with a reducingagent.

FIG. 8D is a view schematically showing the Fab′ antibody in which onereactive thiol group is left by oxidation.

FIG. 8E is a view schematically showing the fluorescently labeled Fab′antibody.

FIG. 8F is a view schematically showing the elecrtophoretic imageobtained by subjecting the fluorescently labeled Fab′ antibody toisoelectric focusing.

FIG. 8G is a view schematically showing the relation of migration timeand fluorescence intensity obtained when electrophoresis is performed tothe immune complex comprising the fluorescently labeled Fab′ antibodyhaving a uniform isoelectric point which is obtained by isoelectricfocusing, and the antigen.

BEST MODE FOR CARRYING OUT THE INVENTION

A method for quantitatively detecting an antigen of the presentinvention comprises (1) a first step of providing an Fab′ antibodyhaving a uniform isoelectric point, said antibody forming an immunecomplex with an antigen in an analytical sample and being modified byadding an amino acid sequence comprising a charged amino acid residueand by being labeled with a fluorescent dye, (2) a second step of mixingthe Fab′ antibody having a uniform isoelectric point with the analyticalsample containing the antigen to obtain a mixture comprising the immunecomplex, (3) a third step of separating the mixture by performingelectrophoresis in a carrier, (4) a fourth step of irradiating anexcitation light which excites the fluorescent dye to the mixtureseparated in the third step to cause fluorescence in the immune complex,and (5) a fifth step of detecting the fluorescence.

In the case of human IgG1 antibody, the antibody has the structure inwhich two polypeptide chains called L chain (light chain) and twopolypeptide chains called H chain (heavy chain) makes a Y-shaped pair,as shown in FIG. 1. Fab′ antibody is a fragment in which a hinge regionor a part thereof is added to an Fab fragment. In the Fab′ antibody, anFd chain (H chain which is in an N-terminal side from a hinge region)consisting of a VH region and a CH1 region, and an L chain consisting ofa VL region and a CL region are bonded by —S—S— bond.

Fab′ antibody used in the present invention is used for detection of anantigen, therefore, it should form an immune complex by reactingspecifically with an antigen included in an analytical sample. The typeof the antigen for detection in the method for quantitative detection ofthe present invention is not limited in particular. Any antigens can beused insofar as they cause an antigen antibody reaction. The type of theanalytical sample is also not limited in particular. Any analyticalsamples can be used insofar as they contain an antigen as an analyte.

An amino acid sequence comprising a charged amino acid residue is addedto the Fab′ antibody used in the present invention. The Fab′ antibodywhich is modified by adding an amino acid sequence comprising a chargedamino acid residue means, in the present invention, the Fab′ antibodywhere an amino acid sequence comprising a positively or negativelycharged amino acid residue is added to at least one site of the Fdchain, the L chain, or the hinge region (or a part thereof) in the Fab′antibody. The positively charged amino acid includes arginine, lysine,and the like. The negatively charged amino acid includes aspartic acid,glutamic acid, and the like. The site of the Fab′ antibody modified withan amino acid sequence comprising a charged amino acid residue is notlimited in particular. However, since the site in the VH region and theVL region of the Fab′ antibody bonds with an antigen, the amino acidsequence comprising a charged amino acid residue is preferably added tothe C-terminal side of the H chain or the L chain in the Fab′ antibodyin a viewpoint of antigen-antibody reactivity. More preferably, it isadded adjacent to a C-terminal of an L chain. The number of amino acidresidues in an amino acid sequence comprising a charged amino acidresidue is not limited as long as it is 1 or more but the number ispreferably 1 to 50. And, the number of charged amino acid residues inthe amino acid sequence is also not limited as long as it is 1 or morebut the number is preferably 1 to 30.

The method of adding the amino acid sequence comprising a charged aminoacid residue to the Fab′ antibody is not limited in particular. Forexample, as described below, polymerase chain reaction (PCR) is firstlyperformed using a primer for adding an amino acid sequence comprising acharged amino acid residue and using a gene expressing an Fab′ antibodyhaving a uniform isoelectric point as a template in order to obtain agene expressing an Fab′ antibody having a uniform isoelectric pointmodified by adding an amino acid sequence comprising a charged aminoacid residue. Then, a host cell transformed with the resultant geneobtained in the above process is cultured. As another method, it ispossible to combine an Fab′ antibody having a uniform isoelectric pointwith an amino acid sequence comprising a charged amino acid residuewhich is prepared independently.

In addition to the modification by the amino acid sequence comprising acharged amino acid residue, the Fab′ antibody used in the presentinvention is modified to show a uniform isoelectric point. The method ofimparting a uniformity of an isoelectric point to the Fab′ antibody isnot limited in particular. However, preferably, a uniformity of anisoelectric point is given by changing an amide group-containing aminoacid (asparagine and/or glutamine) in the CH1 region of the Fab′antibody into an amide group-non-containing amino acid except forcysteine using the genetic engineering, as described below.

The Fab′ antibody used in the present invention is labeled with afluorescent dye in addition to the modification by adding the amino acidsequence comprising a charge modified amino acid residue and byimparting a uniform isoelectric point. The example of the fluorescentdye includes rhodamine, fluorescein, cyanine, indocyanine,indocarbocyanine, pyronine, lucifer yellow, quinacrine, squarillium,coumarin, fluoroanthranilmaleimide, anthracene, and the like. Inparticular, it is preferred that rhodamine and/or cyanine is used. It ismore preferred that rhodamine is used as a fluorescent dye. Rhodaminehas a maximum absorption at 556 nm (molecular extinction coefficient:93,000) in methanol and emits fluorescent light having a maximum of 576nm. Regarding rhodamine, a reference can be made to Handbook ofFluorescent Probes and Research Chemicals, 5th Edition MOLECULAR PROBES,INC., 1992.

Further, in the present invention, an aromatic heterocyclic compound anda polycyclic aromatic hydrocarbon including anthracene, naphthalene,phenanthrene, quinoline, pyrene and perylene can be used as afluorescent dye. Regarding such the fluorescent dye, a reference can bemade to, for example, Fluorescence and Phosphorescence Analysis,authored by Yasuharu Nishikawa and Keizo Hiraki, published by KyoritsuShuppan, 1989.

When above-mentioned fluorescent dye and Fab′ antibody are reacted, thelabeling site with the fluorescent dye on the Fab′ antibody is notlimited in particular. However, the fluorescent dye is preferably boundto a SH group of cysteine residue of the Fab′ antibody. And morepreferably, it is bound to a SH group of cysteine residue which is notinvolved in binding with an L chain and exists in an amino acid sequencewhich adjoins to the C-terminal of the CH1 region of the Fab′ antibody.

When above-mentioned fluorescent dye and Fab′ antibody are reacted, thetype of bond produced by the reaction is not limited in particular,however, preferably, it is at least one bond selected from the groupconsisting of a thioester bond, a dithioester bond, and a thioetherbond. In this case, a fluorescent dye can be directly reacted with afunctional group such as a SH group in an amino acid residue of the Fab′antibody, or can be reacted via a multifunctional compound having atleast one functional group reactive with a functional group (forexample, halogenated methyl group, active ester group, acid chloridegroup, anhydride group, maleimide group and the like) in a fluorescentdye and at least one functional group reactive with a SH group.

In the second step following the first step of the present invention, amixture comprising an immune complex is obtained by mixing theabove-mentioned Fab′ antibody having a uniform isoelectric point whichis modified by adding an amino acid sequence comprising a charged aminoacid residue and is labeled with a fluorescent dye, with an analyticalsample containing the above-mentioned antigen.

In the second step, a method for forming a complex is not particularlylimited. For example, the complex can be formed by the followingprocess. That is, a solution where the analytical sample containing anantigen is dissolved in the ultrapure water or the buffer solution atdesired concentration is mixed with a solution where the Fab′ antibodyhaving a uniform isoelectric point, which is modified by adding an aminoacid sequence comprising a charged amino acid residue and is labeledwith a fluorescent dye (hereinafter, in some cases, it may be called afluorescently labeled charged Fab′ antibody having a uniform isoelectricpoint), is dissolved in the ultrapure water or the buffer solution atdesired concentration. Then, the resultant mixture is kept from a lowtemperature (about 4° C.) to a room temperature (about 25° C.) forseveral minutes to several 10 minutes to obtain the complex. The mixedsolution of the antigen and the fluorescently labeled charged Fab′antibody having a uniform isoelectric point is further dissolved in anelectrophoresis carrier. The different type of an electrophoresiscarrier is used depending on the type of electrophoresis. For example,when the isoelectric focusing is conducted, the electrophoresis carriersuch as a slab gel, a polyacrylamide gel or the like is used for amigration. On the one hand, when the capillary isoelectric focusing isconducted, the ampholite carrier such as Pharmalyte (available fromAmersham Pharmacia Biotech Co.) is used. In addition, when the capillaryisoelectric focusing is conducted, hydroxypropyl methylcellulose or thelike can be added further in order to prevent an electroosmotic flow oran adsorption of protein.

In the third step, electrophoresis of the mixture obtained in the secondstep is performed, and the mixture is separated.

The method for the electrophoresis in the third step is not particularlylimited. Since detection accuracy is excellent, it is preferable toperform the isoelectric focusing. Moreover, since an immune complex canbe detected even with a very small amount, use of the capillaryelectrophoresis is preferable. It is also possible to perform microcellelectrophoresis or microchip electrophoresis. In the present invention,it is more preferable to use the capillary isoelectric focusing becauseit can detect an immune complex with high accuracy even with very smallamount.

When the capillary electrophoresis is performed, the capillary typicallyhas an inside diameter of from several micrometers to about 100micrometers, an outside diameter of several 100 micrometers, and alength of from several 10 centimeters to 100 centimeters, and iscomposed of a soda lime glass or the like. These dimensions, especiallylength, are suitably selected depending on, for example, the type of animmune complex for measurement.

Voltage to be charged is not particularly limited. It can be selectedsuitably according to the type of electrophoresis, the type andconcentration of an immune complex measured, the form and length of amigration carrier, the type of electrophoresis apparatus used, and etc.

In the fourth step, an excitation light which excites the fluorescentdye is irradiated to the mixture separated in the third step to causefluorescence in the immune complex. The type of excitation light is notparticularly limited. When rhodamine is used as a fluorescent dye, useof an argon laser, a semiconductor excitation YAG laser, and a heliumneon laser, or the like is suitable.

In the fifth step, the fluorescence caused in the fourth step isdetected. As a means for fluorescence detection, an optical detectorthat can perform fluorescence detection is used. For example, adensitometer (for example, Shimadzu 2 wave flying-spot scanningdensitometer CS9300PC, available from Shimadzu Co.) is preferably usedas an optical detector.

Fluorescence intensity is measured by an optical detector. Concentrationof a detected complex can be quantified based on a relation betweenconcentration and the fluorescence intensity for the fluorescent dyethat is obtained prior to the measurement.

As explained above, since the antibody used in the present invention hasa uniform isoelectric point, plural peaks observed by electrophoresisare ascribable to the ununiformity of an isoelectric point of theantigen. In addition, since the antibody used in the present inventionis fluorescently labeled with a fluorescent dye or the like, detectioncan be done with high accuracy. Furthermore, since the antibody used inthe present invention is modified by adding an amino acid sequencecomprising a charged amino acid residue, the isoelectric point can bechanged to a desired value while maintaining a uniformity of theisoelectric point by changing the type and/or the amount of introductionof the charged amino acid residue. Therefore, even when an isoelectricpoint of the antigen (analyte) is close to that of the antibody, theimmune complex is detected at the migration time which is different fromthat of the excessive antigen an/or antibody. Then, analysis can be donewith high accuracy.

It is not impossible to chemically modify the Fab′ antibody used in theabove-mentioned method by Shimura K and Karger B L in order to make theisoelectric point of the Fab′ different from that of an antigen byimparting charges. However, since a functional group (for example, aminogroup) used for chemical modification is randomly distributed in theFab′ antibody, uniform modification is impossible. In addition,uniformity of the isoelectric point can be deteriorated by themodification. Therefore, it is impossible with the method by Shimura Kand Karger B L to conduct an analysis with high accuracy when anisoelectric point of the antigen (analyte) is close to that of thefluorescently labeled antibody.

In the present invention, the fluorescently labeled charged Fab′antibody having a uniform isoelectric point which is used for aquantitative detection of the antibody is preferably produced by eitherof the first to fifth method of preparing based on the geneticengineering described below.

The first preparing method based on the genetic engineering comprises(1) a first step of providing an Fd chain gene encoding a VH region, aCH1 region, and an amino acid sequence which adjoins to a C-terminal ofthe CH1 region and comprises a cysteine residue which is not involved inbinding with an L chain in an Fab′ antibody, (2) a second step ofsite-specifically mutating in the Fd chain gene at least one codonencoding an amide group-containing amino acid residue in the CH1 region,into a codon encoding an amide group-non-containing amino acid residueexcept for cysteine to obtain a modified Fd chain gene, (3) a third stepof linking the modified Fd chain gene and an L chain gene encoding an Lchain of the Fab′ antibody in the expressible state to obtain a geneexpressing a modified Fab′ antibody, (4) a fourth step of modifying thegene expressing a modified Fab′ antibody to express an amino acidsequence comprising a charged amino acid residue adjacent to aC-terminal of the L chain to obtain a gene expressing a charge modifiedFab′ antibody, (5) a fifth step of transforming a host cell with thegene expressing a charge modified Fab′ antibody and culturing theresultant transformant to obtain an Fab′ antibody having a uniformisoelectric point, the Fab′ antibody being modified by adding an aminoacid sequence comprising a charged amino acid residue adjacent to theC-terminal of the L chain, and by adding an amino acid sequencecomprising a cysteine residue which is not involved in binding with an Lchain adjacent to the C-terminal of the CH1 region, and (6) a sixth stepof binding a fluorescent dye to the cysteine residue which is notinvolved in binding with an L chain in the Fab′ antibody having auniform isoelectric point obtained in the fifth step.

In the first step, the method of providing the Fd chain gene encodingthe VH region, the CH1 region, and the amino acid sequence which adjoinsto the C-terminal of the CH1 region and comprises a cysteine residuewhich is not involved in binding with the L chain in the Fab′ antibodyis not particularly limited. The gene can be obtained by a methoddescribed below, for example.

That is, after an animal is immunized with an antigen, a monoclonalantibody-producing cell (hybridoma) is prepared according to a methoddescribed in, for example, Antibodies: A Laboratory Manual, Chapter 6,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1988. The wholemRNA is extracted from this monoclonal antibody-producing cell accordingto a protocol described in, for example, BioMag mRNA purification kit(PerSeptive) and this mRNA is used to synthesize a single-stranded cDNA(for example, cDNA Synthesizing System Plus of Amersham PharmaciaBiotech Inc. can be used).

Then, a polymerase chain reaction (PCR) can be performed to obtain an Fdchain gene using this single-stranded cDNA as a template and using a DNAprimer for isolating an Fd chain gene which is designed for introducingan amino acid sequence containing a cysteine residue which is notinvolved in binding with an L chain into a part adjacent to theC-terminal of the CH1 region. Regarding the DNA primer for isolating theFd chain gene, a reference can be made to the base sequence of thenucleic acid of the variable region (V region) and the constant region(C region) which was classified by Kabat et al. (Sequences of Proteinsof Immunological Interest 5th ed., Public Health Service, NIH,Washington D.C., 1991). For designing the primer for introducing theamino acid sequence containing a cysteine residue which is not involvedin binding with an L chain into a part adjacent to a C-terminal of a CH1region, a reference can be made to publications such as Hoogenboom H. R.et al. (Nucleic Acids Res. 1991 Aug. 11; 19(15): 4133–7) and Kang A. S.et al. (Methods (San Diego)(1991), 2(2), 111–18).

The number of amino acid residues of an amino acid sequence to beintroduced into a part adjacent to the C-terminal of the CH1 region isnot limited as long as it is 1 or more but the number is preferably 1 to30. In addition, the number of cysteine residues in the amino acidsequence is not particularly limited but the number is preferably 1 to 3and, more preferably 1.

In addition, upon preparation of a monoclonal antibody-producing cell inthe present invention, the kind of an antigen immunizing an animal andthe kind of an animal to be immunized with the antigen are notparticularly limited. As the antibody gene, those derived from mouse,rat and rabbit can be used. The class and subclass of the antibody arenot particularly limited but it is preferred that the sequence whichconstitutes an IgG antibody is used due to a larger proportion in theall antibodies.

In the second step, at least one codon encoding an amidegroup-containing amino acid residue of the CH1 region issite-specifically mutated into a codon encoding an amidegroup-non-containing amino acid residue except cysteine in the Fd chaingene and this mutating method is not particularly limited. For example,site-specific mutagenesis that is widely used as a method for mutating anucleotide sequence of a gene can be applied thereto. Regarding thesite-specific mutagenesis, for example, a reference can be made toSambrook et al., Molecular Cloning; A Laboratory Manual 2nd Edition,15.2–15.113, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989.

The aforementioned site-specific mutagenesis is preferably conducted bya polymerase chain reaction using a primer for amplifying a CH1 regionwhich substitutes at least one amide group-containing amino acid residueof a CH1 region with an amide group-non containing amino acid residueexcept for cysteine. This primer for amplifying a CH1 region has a basesequence complementary to the base sequence encoding the regioncontaining at least one amide group-containing amino acid residue in theCH1 region of the Fd chain. However, in the base sequence of the primer,at least one codon complementary to a codon encoding the amidegroup-containing amino acid residue is substituted with a codoncomplementary to a codon encoding an amide group-non containing aminoacid except for cysteine.

The aforementioned “amide group-containing amino acid residue” means anamino acid residue having an amide group on its side chain. Anasparagine residue and a glutamine residue are the examples of suchamino acid residue. In the second step, at least one codon encodingthese amino acid residues is site-specifically mutated into a codonencoding an amide group-non-containing amino acid residue except forcysteine. Here, “an amide group-non-containing amino acid residue” meansan amino acid residue having no amide group on its side chain. Eithernatural amino acid residue or unnatural amino acid residue can be usedas the amide group-non-containing amino acid residue. The example of thenatural amino acid residue includes glycine residue, alanine residue,valine residue, leucine residue, isoleucine residue, serine residue,threonine residue, methionine residue, aspartic acid residue, glutamicacid residue, lysine residue, arginine residue, phenylalanine residue,tyrosine residue, proline residue, histidine residue, and tryprophanresidue. As the unnatural amino acid, there are an amino acid in which aside chain of the natural amino acid is substituted with an aromaticring or the like, an artificial amino acid such as a chemicallysynthesized amino acid and the like.

In the present invention, at least one amide group-containing amino acidresidue is preferably mutated into an amide group-non-containing aminoacid residue. When cysteine residues are introduced as the amidegroup-non-containing amino acid residue by the mutation, the resultantamino acid sequence, which contains a lot of cystein residues that arenot involved in binding with the L chain, may show a uniform isoelectricpoint. However, when a fluorescent dye is bound to the cysteine residuesintroduced, an isoelectric point becomes ununiform because the numberand a position of the fluorescent dye become indefinite. For thisreason, in the present invention, it is not preferred to use a cysteineresidue as an amide group-non-containing amino acid residue.

In the present invention, it is preferred that the aforementioned amidegroup-non-containing amino acid residue is an aspartic acid residue, aglutamic acid residue, a glycine residue or a serine residue because ofthe excellent uniformity of isoelectric point of the resultant Fab′antibody having a uniform isoelectric point. In addition, it ispreferred that an Fab′ antibody used has an asparagine residue at the162nd position in the H chain according to the Kabat numbering systemand this asparagine residue is site-specifically mutated into an amidegroup-non-containing amino acid residue except for cysteine. In thepresent invention, it is more preferred that an asparagine residue atthe 162nd position in the H chain according to the Kabat numberingsystem be mutated into an aspartic acid residue. Example of the Fab′antibody having an asparagine residue at the 162nd position in the Hchain according to the Kabat numbering system includes mouse IgGantibody-derived Fab′ antibody and human IgG antibody-derived Fab′antibody. Although a variety of subclasses are present in mouse IgGantibody and human IgG antibody, Fab′ antibodies derived from theseantibodies have an asparagine residue at the 162nd position in the Hchain according to the Kabat numbering system even in a differentsubclass. Here, “amino acid residue at the 162nd position in the H chainaccording to the Kabat numbering system” means that the amino acidresidue in the H chain (Fd chain) of the Fab′ antibody located at“162nd” position based on the method described in Sequence of Proteinsof Immunological Interest (Paperback 5th edition (September 1992)) byElvin A. Kabat. The 162nd amino acid residue in the H chain according tothe Kabat numbering system is in the CH1 region of the Fd chain.

The modified Fd chain gene obtained by the aforementioned second step isbound to the L chain gene encoding the L chain of an Fab′ antibody inthe expressible state in the third step. This can afford a geneexpressing a modified Fab′ antibody.

The L chain gene encoding the L chain of the Fab′ antibody can beobtained according to the similar manner to that for obtaining the Fdgene in the first step. That is, after an animal is immunized with anantigen, a monoclonal antibody-producing cell (hybridoma) is prepared.The whole mRNA is extracted from this monoclonal antibody-producingcell, and a single-stranded cDNA is synthesized using this mRNA. Then,PCR is performed using this cDNA as a template and using a DNA primerfor isolating an L chain gene. In the present invention, the L chaingene can be obtained at the same time with isolating the Fd chain genein the first step. In this case, PCR may be performed using a DNA primerfor isolating the Fd chain gene and the DNA primer for isolating the Lchain gene and using the cDNA as a template in the first step.

In the present invention, in addition to site-specific mutation of atleast one codon encoding an amide group-containing amino acid residue ofthe CH1 region in the Fd chain gene into a codon encoding an amidegroup-non-containing amino acid residue except for cysteine, at leastone codon encoding an amide group-containing amino acid residue(asparagine residue and/or glutamine residue) of the CL region in the Lchain gene obtained as described above may be site-specifically mutatedinto an amide group-non-containing amino acid residue except forcysteine. This site-specific mutation can be performed before or afterbinding with the modified Fd chain gene in the expressible state. Inthis case, it is preferred that the Fab′ antibody used has an asparagineresidue at least one of the 157th, 161st, or 190th positions of the Lchain (all present in a CH region) according to the Kabat numberingsystem. It is preferred that at least one asparagine residue issite-specifically mutated into an amide group-non-containing amino acidresidue except for cysteine. In particular, it is preferred that anasparagine residue at the 161st position in the L chain according to theKabat numbering system is site-specifically mutated into an amidegroup-non-containing amino acid residue except for cysteine. It is morepreferred that an asparagine residue at the 161st position in the Lchain is mutated into an aspartic acid residue. Regarding thesite-specific mutating method, a reference can be made to Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd Edition, 15.2–15.113,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989 as describedabove. As in the case of the CH1 region, it is preferred that thesite-specific mutation of the CL region is performed by a polymerasechain reaction using a primer for amplifying the CL region whichsubstitutes at least one amide group-containing amino acid residue ofthe CL region with an amide group-non-containing amino acid residueexcept for cysteine.

By binding above-obtained L chain gene and the aforementioned modifiedFd chain gene via a linker base sequence or the like, a gene expressinga modified Fab′ antibody including above genes can be obtained. Moreparticularly, the L chain gene, the modified Fd chain gene and thelinker base sequence are purified using, for example, low melting pointagarose gel electrophoresis and purified genes are digested by anappropriate restriction enzyme. Then, digested genes are ligated so thatthe modified Fd chain gene, the linker base sequence and the L chaingene are arranged in this order. The linker base sequence can beobtained using a template plasmid vector for expressing a protein andusing a DNA primer for isolating the linker base sequence.

In the fourth step following the third step, the gene expressing amodified Fab′ antibody is modified to express an amino acid sequencecomprising a charged amino acid residue adjacent to a C-terminal of theL chain. This can afford a gene expressing a charge modified Fab′antibody. The method for this modification is not particularly limited.For example, it can be obtained by performing PCR using the geneexpressing a modified Fab′ antibody as a template and using a primer foradding an amino acid sequence comprising a charged amino acid residueadjacent to the C-terminal of the L chain.

In the fifth step, a host cell is transformed with the gene expressing acharge modified Fab′ antibody and culturing the resultant transformant.This can afford an Fab′ antibody having a uniform isoelectric point, theFab′ antibody being modified by adding an amino acid sequence comprisinga charged amino acid residue adjacent to the C-terminal of the L chain,and by adding an amino acid sequence comprising a cysteine residue whichis not involved in binding with an L chain adjacent to the C-terminal ofthe CH1 region

That is, the gene expressing a charge modified Fab′ antibody obtained inthe fourth step is ligated to an appropriate vector and this isintroduced into a host cell to transform it. As a vector, a variety ofknown vectors such as a vector derived from a plasmid, a phage, a cosmidor the like can be used. As a host cell, for example, there areprocaryotic cell, that is, Escherichia coli (SOLR, JM109, XL1-BlueMRF′,BL21 (DE3), HB2151), Bacillus subtilis, Bacillus brevis, and eukaryoticcell, that is, yeast, and cell derived from an animal (HB101, CHO cell,COS cell, COP-5, C127, 3T3 cell and the like). As a host cell, it ispreferred that a protease-non-producing bacteria is used from aviewpoint of degradation resistance of the Fab′ antibody having auniform isoelectric point.

As the method of introducing the vector into the host cell, knownmethods including a microinjection method, electropolation method andthe like can be applied. The method for culturing the transformant isnot particularly limited and a medium suitable for culturing thetransformant may be selected. In addition, as the method for extractingthe Fab′ antibody having a uniform isoelectric point produced byculturing a transformant, cell homogenization method, lysis of cell wallusing a surfactant such as SDS or an enzyme, ultrasonicating method, andthe like can be adopted. The method for purifying the extracted Fab′antibody having a uniform isoelectric point includes centrifugation suchas ultracentrifugation and gradient centrifugation, column-separationutilizing an affinity column and the like, gel-separation utilizingpolyacrylamide gel, and the like.

In the sixth step, a fluorescent dye is bound to the cysteine residuewhich is not involved in binding with an L chain in the Fab′ antibodyhaving a uniform isoelectric point obtained in the fifth step.

Here, the fluorescent dye described above can be used. The type of bondproduced by the reaction is preferably at least one bond selected fromthe group consisting of a thioester bond, a dithioester bond, and athioether bond. In this case, the fluorescent dye can be directlyreacted with a functional group such as a SH group in the amino acidresidue of the Fab′ antibody, or can be reacted via a multifunctionalcompound having at least one functional group reactive with a functionalgroup in the fluorescent dye and at least one functional group reactivewith a SH group.

In the first preparing method based on the genetic engineering describedabove, after the modified Fd chain gene and the L chain gene are linkedin the expressible state in the third step, the linked gene is modifiedin the fourth step to express an amino acid sequence comprising acharged amino acid residue adjacent to a C-terminal of the L chain. Asanother preparing method, the L chain gene can be modified to express anamino acid sequence comprising a charged amino acid residue adjacent tothe C-terminal of the L chain before the modified Fd chain gene and theL chain gene are linked. This preparing method is called the secondpreparing method based on the genetic engineering, hereinafter.

That is, in the second preparing method based on the geneticengineering, in the first step there is provided an Fd chain geneencoding a VH region, a CH1 region, and an amino acid sequence whichadjoins to a C-terminal of the CH1 region and comprises a cysteineresidue which is not involved in binding with an L chain in an Fab′antibody. In the second step, at least one codon encoding an amidegroup-containing amino acid residue in the CH1 region of the Fd chaingene is site-specifically mutated into a codon encoding an amidegroup-non-containing amino acid residue except for cysteine to obtain amodified Fd chain gene.

This first step and second step can be performed similarly to the firststep and second step of the first preparing method based on the geneticengineering. The preferred number of an amino acid residue in the aminoacid sequence introduced adjacent to the C-terminal of the CH1 region,the preferred number of a cysteine residue in the amino acid sequence,and the type of antigen and antibody are the same as those described inthe first preparing method based on the genetic engineering. The kind ofa amide group-containing amino acid residue, the kind of an amidegroup-non-containing amino acid residue, and the preferred amino acidresidue thereof are the same as those described in the second step ofthe first preparing method based on the genetic engineering.

In the third step, an L chain gene encoding an L chain of the Fab′antibody is provided. The L chain gene can be obtained according to thesimilar manner to that described in the third step of the firstpreparing method based on the genetic engineering. In addition, the Lchain gene can be also obtained at the same time with isolating the Fdchain gene in the first step.

In the fourth step following the third step, the L chain gene ismodified to express an amino acid sequence comprising a charged aminoacid residue adjacent to the C-terminal of the L chain. This can afforda charge modified L chain gene. The modification in this step can bedone according to the similar manner to that described in the fourthstep of the first preparing method based on the genetic engineering.Here, at least one codon encoding an amide group-containing amino acidresidue of the CL region in the L chain gene may be site-specificallymutated into a codon encoding an amide group-non-containing amino acidresidue except for cysteine. This site-specific mutation can beperformed in the third step.

In the fifth step, the modified Fd chain gene and the charge modified Lchain gene are liked in the expressible state. This can afford a geneexpressing a charge modified Fab′ antibody. Conditions for linking inthe expressible state are the same as those described in the third stepof the first preparing method based on the genetic engineering.

Subsequently, in the sixth step, a host cell is transformed with thegene expressing a charge modified Fab′ antibody and culturing theresultant transformant to obtain an Fab′ antibody having a uniformisoelectric point, the Fab′ antibody being modified by adding an aminoacid sequence comprising a charged amino acid residue adjacent to theC-terminal of the L chain, and by adding an amino acid sequencecomprising a cysteine residue which is not involved in binding with an Lchain adjacent to the C-terminal of the CH1 region. In the seventh step,a fluorescent dye is bound to the cysteine residue which is not involvedin binding with the L chain in the Fab′ antibody having a uniformisoelectric point. The sixth and seventh steps can be performedsimilarly to the fifth and sixth steps of the above-mentioned firstpreparing method based on the genetic engineering.

In the first and second preparing methods based on the geneticengineering described above, after the Fd chain gene issite-specifically mutated, the resultant gene is linked with an L chaingene. As the third preparing method based on the genetic engineering,after the Fd chain gene and the L chain gene are linked, the Fd chaingene can be site-specifically mutated.

That is, in the first step, there is provided an Fd chain gene encodinga VH region, a CH1 region, and an amino acid sequence which adjoins tothe C-terminal of the CH1 region and comprises a cysteine residue whichis not involved in binding with an L chain in an Fab′ antibody, andprovided an L chain gene encoding the L chain of the Fab′ antibody. Inthe second step following the first step, the Fd chain gene and the Lchain gene are linked in the expressible state to obtain a geneexpressing an Fab′ antibody. In this case, the Fd chain gene can beobtained according to the similar manner to that described in the firststep of the first preparing method based on the genetic engineering. TheL chain gene can be obtained according to the similar manner to thatdescribed in the third step of the first preparing method based on thegenetic engineering. Linking the Fd chain gene and the L chain gene inthe expressible state can be done according to the similar manner tothat described in the third step of the first preparing method based onthe genetic engineering. That is, a linker base sequence is obtainedusing a template plasmid vector for expressing a protein and using a DNAprimer for isolating a linker base sequence. This linker base sequence,the Fd chain gene and the L chain gene are ligated in the expressiblestate. The preferred number of an amino acid residue in the amino acidsequence introduced adjacent to the C-terminal of the CH1 region, andthe suitable number of a cysteine residue in the amino acid sequence arethe same as those described in the first preparing method based on thegenetic engineering.

In the third step, the gene expressing an Fab′ antibody is modified toexpress an amino acid sequence comprising a charged amino acid residueadjacent to the C-terminal of the L chain, and in the gene expressing anFab′ antibody at least one codon encoding an amide group-containingamino acid residue in the CH1 region is site-specifically mutated into acodon encoding an amide group-non-containing amino acid residue exceptfor cysteine. This can afford a gene expressing a charge modified Fab′antibody.

In this case, there is no limitation of the order of the modificationsdescribed above—modifation to express an amino acid sequence comprisinga charged amino acid residue adjacent to the C-terminal of the L chain,and site-specific mutation of at least one codon encoding an amidegroup-containing amino acid residue in the CH1 region into a codonencoding an amide group-non-containing amino acid residue except forcysteine.

For example, firstly, the gene expressing an Fab′ antibody is modifiedto express an amino acid sequence comprising a charged amino acidresidue adjacent to a C-terminal of the L chain to obtain a geneexpressing an Fab′ antibody having a charged amino acid. Then, at leastone codon encoding an amide group-containing amino acid residue in theCH1 region in the gene expressing an Fab′ antibody having a chargedamino acid is site-specifically mutated into a codon encoding an amidegroup-non-containing amino acid residue except for cysteine to obtain agene expressing a charge modified Fab′ antibody. Alternatively, firstly,at least one codon encoding an amide group-containing amino acid residuein the gene expressing an Fab′ antibody is site-specifically mutatedinto a codon encoding an amide group-non-containing amino acid residueexcept for cysteine to obtain a gene expressing a modified Fab′antibody. Then, the gene expressing a modified Fab′ antibody is modifiedto express an amino acid sequence comprising a charged amino acidresidue adjacent to the C-terminal of the L chain to obtain a geneexpressing a charge modified Fab′ antibody.

Site-specific mutation in the third step can be performed according tothe similar manner to that described in the second step of the firstpreparing method based on the genetic engineering. The preferred amidegroup-containing amino acid, the preferred amide group-non-containingamino acid, and the preferred amino acid thereof are the same as thosedescribed in the first preparing method based on the geneticengineering. Modification to express an amino acid sequence comprising acharged amino acid residue adjacent to the C-terminal of the L chain canbe done according to the similar manner to that described in the fourthstep of the first preparing method based on the genetic engineering.Here, at least one codon encoding an amide group-containing amino acidresidue of the CL region in the gene expressing Fab′ antibody may besite-specifically mutated into a codon encoding an amidegroup-non-containing amino acid residue except for cysteine. Thissite-specific mutation can be performed in the first step.

In the fourth step, a host cell is transformed with the gene expressinga charge modified Fab′ antibody and culturing the resultant transformantto obtain an Fab′ antibody having a uniform isoelectric point, the Fab′antibody being modified by adding an amino acid sequence comprising acharged amino acid residue adjacent to the C-terminal of the L chain,and by adding an amino acid sequence comprising a cysteine residue whichis not involved in binding with an L chain adjacent to the C-terminal ofthe CH1 region. The kind of a host cell, the kind of a vector which isintroduced into a host cell, the method for introducing a vector into ahost cell and the method for extracting an Fab′ antibody having auniform isoelectric point produced by culturing a transformant are thesame as those described in the fifth step of the first preparing methodbased on the genetic engineering.

In the fifth step, a fluorescent dye is bound to the cysteine residuewhich is not involved in binding with the L chain in the Fab′ antibodyhaving a uniform isoelectric point obtained in the fourth step. The kindof fluorescent dye, the preferred fluorescent dye, and the kind ofbonding between cysteine residue and fluorescent dye are the same asthose described in the sixth step of the first preparing method based onthe genetic engineering.

In addition to the above-mentioned first to third preparing method basedon the genetic engineering, the fourth preparing method based on thegenetic engineering described below is also applicable.

That is, in the first step, there is provided a CH1 gene encoding a CH1region and an amino acid sequence which adjoins to the C-terminal of theCH1 region and comprises a cysteine residue which is not involved inbinding with the L chain in the first Fab′ antibody, and a CL geneencoding the CL region of the first Fab′ antibody.

The CH1 gene in the first step can be obtained, for example, byextracting the whole mRNA from a monoclonal antibody-producing cell(hybridoma), synthesizing a single-stranded cDNA using this mRNA, andperforming PCR using this cDNA as a template and using a primer tointroduce an amino acid sequence comprising a cysteine residue which isnot involved in binding with the L chain into a part adjacent to theC-terminal of the CH1 region. The CH1 gene may be any one that encodes aregion containing the CH1 region. The CH1 gene can be the one encodingonly the CH1 region or the one encoding the CH1 region and the VHregion. In addition, the CH1 gene may be the one encoding the CH1 regionand the hinge region, or the one encoding the CH1 region, the VH regionand the hinge region. Regarding the VH region and the hinge region, atleast part thereof may be encoded. The preferred number of an amino acidresidue in the amino acid sequence introduced adjacent to the C-terminalof the CH1 region, the preferred number of a cysteine residue in theamino acid sequence, and the type of antigen and antibody are the sameas those described in the first preparing method based on the geneticengineering.

The CL gene in the first step can be obtained at the same time orindependently with obtaining the above-mentioned CH1 gene. It can beobtained by performing PCR using a single-stranded cDNA, which isobtained from a mRNA derived from a monoclonal antibody-producing cell,as a template, and using a primer for isolating a CL chain gene

Here, at least one codon encoding an amide group-containing amino acidresidue in the CL gene obtained in the first step may besite-specifically mutated into a codon encoding an amidegroup-non-containing amino acid residue except for cysteine.

In the second step, in the CH1 gene, at least one codon encoding anamide group-containing amino acid residue in the CH1 region issite-specifically mutated into a codon encoding an amidegroup-non-containing amino acid residue except for cysteine. This canafford a modified CH1 gene.

This site-specific mutation can be done according to the similar mannerto that described in the second step of the first preparing method basedon the genetic engineering. The kind of a amide group-containing aminoacid residue, the kind of an amide group-non-containing amino acidresidue, and the preferred amino acid residue thereof are the same asthose described in the first preparing method based on the geneticengineering.

Subsequently, in the third step, the modified CH1 gene is cut with arestriction enzyme to obtain a gene fragment encoding the CH1 region.The restriction enzyme used herein is not particularly limited. Theexample of the restriction enzyme includes BamHI and BglI.

In the fourth step, there is provided a VH gene encoding the VH regionof the second Fab′ antibody and a VL gene encoding the VL region of thesecond Fab′ antibody. Here, the VH gene and the VL gene of the secondFab′ antibody can be obtained by the following process, for example.That is, a single-stranded cDNA is synthesized using the mRNA of theantibody from which the second Fab′ antibody is derived, then, PCR isperformed using the cDNA as a template and using a DNA primer forisolating the VH gene and the VL gene. The second Fab′ antibody may bethe same as or different from the first Fab′ antibody in terms of theclass and the subclass. In addition, the animal species from which thesecond Fab′ antibody is derived may be the same as or different fromthose of the first Fab′ antibody. For example, the second Fab′ antibodymay be human IgG antibody and the first Fab′ antibody may be mouse IgGantibody.

In the fifth step, the gene fragment, the CL gene, the VH gene and theVL gene are linked in the expressible state to obtain a gene expressinga modified Fab′ antibody. Linking in the expressible state can beperformed according to the similar manner to that described in thirdstep of the first preparing method based on the genetic engineering.

In the sixth step, the gene expressing a modified Fab′ antibody ismodified to express an amino acid sequence comprising a charged aminoacid residue adjacent to the C-terminal of the CL region. This canafford a gene expressing a charge modified Fab′ antibody. Modificationin the sixth step can be done according to the similar manner to thatdescribed in fourth step of the first preparing method based on thegenetic engineering.

Subsequently, in the seventh step, a host cell is transformed with thegene expressing a charge modified Fab′ antibody and the resultanttransformant is cultured to obtain an Fab′ antibody having a uniformisoelectric point. In the Fab′ antibody having a uniform isoelectricpoint obtained in the seventh step, an amino acid sequence comprising acharged amino acid residue is added adjacent to the C-terminal of the Lchain, and an amino acid sequence comprising a cysteine residue which isnot involved in binding with an L chain is added adjacent to theC-terminal of the CH1 region.

The kind of a host cell, the kind of a vector to be introduced into ahost cell, the method for introducing the vector into the host cell, andthe method for extracting an Fab′ antibody having a uniform isoelectricpoint produced by culturing the transformant are the same as thosedescribed in the fifth step of the first preparing method based on thegenetic engineering.

In the eighth step, a fluorescent dye is bound to the cysteine residuewhich is not involved in binding with the L chain in the Fab′ antibodyhaving a uniform isoelectric point obtained in the seventh step. Thekind of fluorescent dye, the preferred fluorescent dye, and the kind ofbonding between the cysteine residue and the fluorescent dye are thesame as those described in the sixth step of the first preparing methodbased on the genetic engineering.

In the fourth preparing method based on the genetic engineeringdescribed above, the gene fragment of the modified CH1 gene, the CLgene, the VH gene and the VL gene in the expressible state are linked inthe fifth step, then, the linked gene is modified to express an aminoacid sequence comprising a charged amino acid residue adjacent to theC-terminal of the CL region in the sixth step. As another preparingmethod, the CL gene can be modified to express an amino acid sequencecomprising a charged amino acid residue adjacent to the C-terminal ofthe L chain before the CL gene is linked with other genes (or a genefragment). This preparing method is called the fifth preparing methodbased on the genetic engineering, hereinafter.

That is, in the fifth preparing method based on the genetic engineering,there is provided in the first step a CH1 gene encoding a CH1 region andan amino acid sequence which adjoins to a C-terminal of the CH1 regionand comprises a cysteine residue which is not involved in binding withan L chain in a first Fab′ antibody, and a CL gene encoding a CL regionof the first Fab′ antibody. In the second step, at least one codonencoding an amide group-containing amino acid residue in the CH1 regionis site-specifically mutatated into a codon encoding an amidegroup-non-containing amino acid residue except for cysteine to obtain amodified CH1 gene. Then, in the third step, the modified CH1 gene is cutwith a restriction enzyme to obtain a gene fragment encoding the CH1region.

The first to third steps of the fifth preparing method based on thegenetic engineering can be performed according to the similar manner tothose described in the first to third steps of the fourth preparingmethod based on the genetic engineering. Here, at least one codonencoding an amide group-containing amino acid residue in the CL geneobtained in the first step may be site-specifically mutated into a codonencoding an amide group-non-containing amino acid residue except forcysteine.

Subsequently, in the fourth step, the CL gene is modified to express anamino acid sequence comprising a charged amino acid residue adjacent tothe C-terminal of the CL region to obtain a charge modified CL gene.Modification in the fourth step can be done according to the similarmanner to that described in fourth step of the first preparing methodbased on the genetic engineering. The fourth step can be performedbefore the second step.

Subsequently, in the fifth step, there is provided a VH gene encodingthe VH region of the second Fab′ antibody and a VL gene encoding the VLregion of the second Fab′ antibody. In the sixth step, the genefragment, the charge modified CL gene, the VH gene and the VL gene arelinked in the expressible state to obtain a gene expressing a chargemodified Fab′ antibody.

The step for providing a VH gene and a VL gene can be performed in thesimilar manner to that described in the fourth step of the fourthpreparing method based on the genetic engineering. Linking in theexpressible state can be performed according to the similar manner tothat described in third step of the first preparing method based on thegenetic engineering.

Subsequently, in the seventh step, a host cell is transformed with thegene expressing a charge modified Fab′ antibody and the resultanttransformant is cultured to obtain an Fab′ antibody having a uniformisoelectric point. In the Fab′ antibody having a uniform isoelectricpoint obtained in the seventh step, an amino acid sequence comprising acharged amino acid residue is added adjacent to the C-terminal of the Lchain, and an amino acid sequence comprising a cysteine residue which isnot involved in binding with an L chain is added adjacent to theC-terminal of the CH1 region

The kind of a host cell, the kind of a vector to be introduced into ahost cell, the method for introducing the vector into the host cell andthe method for extracting an Fab′ antibody having a uniform isoelectricpoint produced by culturing a transformant are the same as thosedescribed in the fifth step of the first preparing method based on thegenetic engineering.

In the eighth step, a fluorescent dye is bound to the cysteine residuewhich is not involved in binding with an L chain in the Fab′ antibodyhaving a uniform isoelectric point obtained in the seventh step. Thekind of fluorescent dye, the preferred fluorescent dye, and the kind ofbonding between the cysteine residue and the fluorescent dye are thesame as those described in the sixth step of the first preparing methodbased on the genetic engineering.

EXAMPLES

The present invention will be explained in detail by way of preferredExample but is not limited thereto. In addition, in Example, unlessindicated for genetic-engineering procedures, the procedures wereaccording to Sambrook et al., Molecular Cloning: A Laboratory Manual 2ndEdition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989. Inaddition, reagents which are not particularly indicated were purchasedfrom Takara Shuzo or Wako Pure Chemical Industries Ltd. for use.

Abbreviations used in this Example are as follows;

PCR: polymerase chain reaction (Gene amplifying method)

BAP: bacterial alkaline phosphatase

IPTG: isopropyl-β-D-thiogalactopyranoside

PBS: phosphate buffered saline

BSA: bovine serum albumin

(1) Establishment of a Hybridoma

A hybridoma producing anti-human alpha-1-antitrypsin antibody was madeusing human alpha-1-antitrypsin (manufactured by Carbiochem-Noviochem)as an immunization antigen according to the following method:

A BALB/c mouse was immunized four times with the above immunogen, spleencells were taken, cell fusion was performed using a cultured mousemarrow cell (x63Ag8) and polyethylene glycol and cloning was performed.The binding activity of the immunogen with the antibody in the culturesupernatant of the resultant clone was measured by the enzyme-antibodymethod A clone that was considered to be positively reactive was furtherconfirmed using the indirect fluorescent method. Then, nine kinds ofhybridomas that produce the alpha-1-antitrypsin antibody wereestablished. The antibodies produced by these hybridomas are the onesthat bind to the human alpha-1-antitrypsin. For preparing the Fd geneand the L chain (κ chain) gene of the Fab′ antibody having a uniformisoelectric point described below, these cells that produce anti-humanalpha-1-antitrypsin antibody having the anti-alpha-1-antitrypsinactivity were used.

A gene expressing anti-human alpha-1-antitrypsin Fab′ antibody wasisolated from a hybridoma producing an IgG1 antibody against humanalpha-1-antitrypsin as follows:

That is, the total RNA was extracted from the cells producing theanti-human alpha-1-antitrypsin Fab′ antibody according to the protocolof BioMag mRNA purification kit (PerSeptive), and a single-stranded cDNAwas synthesized using cDNA Synthesis System Plus (manufactured byAmersham Pharmacia Biotech Inc.). Polymerase chain reaction (PCR) wasperformed using the aforementioned cDNA as a template and using a DNAprimer for isolating the Fd chain gene and a DNA primer for isolatingthe L chain gene, which were synthesized based on the base sequence ofthe variable region (V region) and the constant region (C region)classified by Kabat et al. (Sequences of Proteins of ImmunologicalInterest, 5^(th) ed., Public Health Service, NIH, Washington D.C.,1991). Here, for designing a primer, reference was made to Hoogenboom H.R. et al. (Nucleic Acids Res., 1991, Aug. 11:19 (15): 4133–7), and KangA. S. et al. (Methods (San Diego) (1991), 2 (2), 111–18).

In order to express an antibody which binds to the humanalpha-1-antitrypsin as an Fab′ antibody, a DNA primer was designed sothat both the heavy chain (H chain) and the light chain (L chain)contain constant region. That is, a 5′ primer (F5-1 primer shown below)and a 3′ primer (F3 primer shown below) were designed as a DNA primerfor isolating the Fd chain gene, and a 5′ primer (Kapper5 primer shownbelow) and a 3′ primer (K3-1 primer shown below) were designed as a DNAprimer for isolating the L chain gene.

The F5-1 primer which is a 5′ primer for isolating the Fd chain gene hadthe sequence shown below (SEQ ID NO: 1) and the F3 primer which is a 3,primer for isolating the Fd chain gene had the sequence shown below (SEQID NO: 2). In the following sequences, 5, and 3′ mean a 5′ side and a 3,side, respectively. S indicates C or G, M indicates A or C, R indicatesA or G, and W indicates A or T.

F5-1 primer (SEQ ID NO: 1) 5′ SAGGTSMARCTGCAGSAGTCWGG 3′ F3 primer (SEQID NO: 2) 5′ GCGTCATCTAGAACAACCACAATCCCTGGGCACA 3′

The F3 primer was designed so that a base sequence comprising a cysteineresidue which is not involved in disulfide bond with the L chain can beintroduced into a part adjacent to the C-terminal of the CH1 region, andin order to ligate to the L chain via a linker, it was designed so thata Xba I site is added thereto.

The Kapper5 primer which is a 5′ primer for isolating the L chain genehad the sequence shown below (SEQ ID NO: 3) and the K3-1 primer which isa 3′ primer for isolating the L chain gene had the sequence shown below(SEQ ID NO: 4). W indicates A or T, S indicates C or G, B indicates abase other than A, N indicates A, T, G or C, M indicates A or C, Dindicates a base other than C, Y indicates C or T, H indicates a baseother than G, respectively.

Kapper5 primer (SEQ ID NO: 3): 5′ CCAGWTSYGAGCTCSWBNTSACNCAGNMDYCH 3′K5-1 primer (SEQ ID NO: 4): 5′ ACACTCATTCCTGTTGAAGCT 3′

PCR was performed under the conditions of 30 cycles of 1 minute at 94°C., 1 minute at 55° C., and 1 minute at 72° C. After PCR, DNA fragmentsof the resultant Fd chain, the linker base sequence and the L chain (κchain) were purified by agarose gel electrophoresis, the Fd chain wasdigested with XbaI, the linker base sequence with XbaI and SacI, and theL chain (κ chain) with SacI. Respective DNA fragments were ligated sothat the Fd chain, the linker base sequence and the L chain (κ chain)were arranged in this order. The ligation product was extracted withphenol/chloroform/isoamylalcohol (25/24/1). This was dissolved in TEbuffer, and PCR was performed again with a primer designed so that aSfiI site is added to a 5′ side of the Fd chain and a NotI site is addedto a 3′ side of the L chain. PCR was performed for 25 cycles of 1 minuteat 94° C., 1 minute at 55° C. and 2.5 minutes at 72° C.

After the resultant PCR amplified-product was purified, it was digestedwith SfiI (20 U per reaction) at 50° C. for 4 hours and with NotI (40 Uper reaction) at 37° C. for 4 hours. This was cloned into the pCANTAB5Eplasmid vector (manufactured by Amersham Pharmacia Biotech Inc.) (seeFIGS. 2A and B). The pCANTAB plasmid vector secretes a protein derivedfrom the gene outside a periplasm of Escherichia coli and it contains asignal peptide which expresses a gene incorporated into a vector. Theprocedures and methods for constructing a vector which areconventionally used in the genetic-engineering field can be used.

(3) Transformation for Expressing Anti-Human Alpha-1-Anititrypsin Fab′Antibody

The plasmid expressing an anti-human alpha-1-anititrypsin Fab′ antibody,that was made by cloning a gene expressing an anti-humanalpha-1-anititrypsin Fab′ antibody was cloned into the pCANTAB5E plasmidvector, was transformed into the commercially available Escherichia coliHB2151 (manufactured by Amersham Pharmacia Biotech Inc.). TheEscherichia coli HB2151 was made competent according to the protocol ofExpression Module/Recombinant Pharge Antibody System (manufactured byAmersham Pharmacia Biotech Inc.). The transformed Escherichia coliHB2151 was seeded on the SOBAG medium and incubated at 30° C. overnight.Colony lift assay was performed on the produced colony according to theprotocol of HRP/Anti-E tag Conjugate (manufactured by Amersham PharmaciaBiotech Inc.) to screen bacteria expressing Fab′ antibody which causesan antigen-antibody reaction against the human alpha-1-anititrypsin.

A plurality of screened bacteria expressing Fab′ antibody was selected,seeded on the 2YT-AG medium and cultured by shaking at 30° C. overnightaccording to the protocol of Expression Module/Recombinant PhargeAntibody System (manufactured by Amersham Pharmacia Biotech Inc.). Theshaken cultured culture was added to 10-fold amount of the 2YT-AG mediumand cultured by shaking at 30° C. until A600 became 0.5. The bacteriawere collected by centrifugation at a room temperature and thesupernatant was removed. The bacteria were suspended in the same amountof 2YT-AI (no glucose, 100 μg/ml ampicillin, 1 mM IPTG), and cultured at30° C. overnight to induce an antibody. Then, the bacteria wereprecipitated by centrifugation, and the supernatant was taken. 100 μl ofthe culture supernatant containing anti-human alpha-1-anititrypsin Fab′antibody induced by IPTG was added to a microtiterplate for adsorbing anantigen on which the human alpha-1-anititrypsin (manufactured byCarbiochem-Novabiochem) was fixed. Then, ELISA was performed accordingto the protocol of HRP/Anti-E tag Conjugate (manufactured by AmershamPharmacia Biotech Inc.) to screen bacteria expressing anti-humanalpha-1-anititrypsin Fab′ antibody.

(4) Transformation for Base Sequence Determination and Sequencing of anAntibody Gene

A plasmid DNA was extracted from bacteria expressing anti-humanalpha-1-anititrypsin Fab′ antibody obtained from screening and it wastransformed into the commercially available Escherichia coli XL10-GOLD(manufactured by Stratagene) for sequencing. XL10-GOLD transformed withthe gene expressing anti-human alpha-1-anititrypsin Fab′ antibody wasmixed with a DNA (about 10 ng) of a vector expressing the antibody geneand stored in ice for 30 minutes according to Epicurian Coli XL10-Goldultracompetent Cells (manufactured by Stratagene). Then, afterheat-treatment at 42° C. for 30 seconds, 900 μL of NZY medium (NZ amine10 g, yeast extract 5 g, sodium chloride 5 g, magnesium chloride 12.5mM, magnesium sulfate 12.5 mM, glucose 20 mM:, pH 7.5: per 1 liter) wasadded and cultured by shaking at 37° C. for about 1 hour. The culturewas spread on LB agar medium containing 50 μg/ml ampicillin (tryptone 10g, yeast extract 5 g, sodium chloride 5 g, agar 15 g [pH 7]: per 1liter) to select the transformed resistant strain containing geneexpressing anti-human alpha-1-anititrypsin Fab′ antibody.

The pCANTAB5E plasmid containing gene expressing anti-humanalpha-1-anititrypsin Fab′ antibody was extracted from the selectedresistant strain and the base sequence of each part of the geneexpressing anti-human alpha-1-anititrypsin Fab′ antibody was determinedby a chain terminator method using dideoxynucleotides (manufactured byPerkin Elmer) to find that an expressible open reading frame (ORF) wastaken. In addition, it was confirmed that the isolated gene expressinganti-human alpha-1-anititrypsin Fab′ antibody contained the Fd chaingene (gene of the VH region and the CH1 region) and the L chain gene(gene of the VL region and the CL region).

(5) Induction and Purification of an Anti-Human Alpha-1-AnititrypsinFab′ Antibody Produced by Escherichia coli

In order to use an anti-human alpha-1-anititrypsin Fab′ antibodyproduced by Escherichia coli in an experiment described below, antibodywas induced in a large scale using the screened cell strain and theresultant product was purified. That is, induction and purification ofan anti-human alpha-1-anititrypsin Fab′ antibody were performedaccording to the protocol of RPAS Purification Module (manufactured byAmersham Pharmacia Biotech Inc.).

A single colony was picked up from the screened Escherichia coli HB2151strain containing the gene expressing an anti-human alpha-1-anititrypsinFab′ antibody, seeded on 2YT-AG medium, then, cultured at 30° C.overnight according to the protocol Expression Module/Recombinant PhargeAntibody System (manufactured by Amersham Pharmacia Biotech Inc.). Theshaken cultured culture was added to 10-fold amount of 2YT-AG medium andcultured by shaking at 30° C. until A600 became 0.5. The bacteria werecollected by centrifugation and the supernatant was removed. Thebacteria were suspended in the same amount of 2YT-AI (containing noglucose) and cultured at 30° C. overnight. The bacteria wereprecipitated by centrifugation, the supernatant was taken, filtered with0.45 μm filter (manufactured by Millipore), and pH was adjusted to 7 toobtain a culture supernatant.

The culture supernatant containing anti-human alpha-1-anititrypsin Fab′antibody induced by IPTG was bound to anti E-tag affinity column at aflow rate of 5 ml/minutes. 25 ml of Binding buffer attached (rate of 5ml/min.) was flown to wash out the culture supernatant containing noantibody and anti-human alpha-1-anititrypsin Fab′ antibody produced byEscherichia coli was eluted (rate of 5 ml/min.) with 10 ml of Elutionbuffer. The eluted anti-human alpha-1-anititrypsin Fab′ antibody wasneutralized by immediately adding 1/10 amount (relative to Elutionbuffer) of Neutralizing buffer. Purification was conducted by affinitychromatography using anti E-tag antibody as a ligand. The neutralizedanti-human alpha-1-anititrypsin Fab′ antibody was concentrated usingMicrocon (for fraction: molecular weight 30000) (manufactured byMillipore), dissolved in 1 ml of PSB buffer, and stored at −80° C.

(6) Fluorescently Labeling of Anti-Human Alpha-1-Anititrypsin Fab′Antibody Produced by Escherichia coli

Preparation of tetramethylrhodamine-5-iodoacetamide which is afluorescent dye (fluorescent labeling agent) was performed as follows:That is, 1 mg of tetramethylrhodamine-5-iodoacetamide (manufactured byMolecular Probes) was dissolved in 0.6 ml of 50% acetonitrile, and thesolution was centrifuged at 10,000 rpm for 5 minutes to remove theprecipitates. The supernatant was subjected to a reverse chromatographycolumn equilibrated with 25% acetonitrile-0.1% trifluoroacetic acid(Toso-ODS-80Ts, diameter 4.6 mm, length 25 cm), and eluted with a lineargradient of 25 to 55% acetonitrile over 30 minutes to detect bymonitoring the absorbance at 280 nm. The greatest peak was taken, andthe concentration thereof was determined by absorbance measurement usingmolecular extinction coefficient at 543 nm of 87,000. This was used forfluorescently labeling the purified anti-human alpha-1-anititrypsin Fab′antibody as purified tetramethylrhodamine-5-iodoacetamide.

The purified anti-human alpha-1-anititrypsin Fab′ antibody wasfluorescently labeled as follows: That is, a concentrated solution ofanti-human alpha-1-anititrypsin Fab′ antibody (100 μl) was diluted with10-fold amount of a 0.1M phosphate buffer containing 5 mM EDTA (pH 7.0)and centrifuged with Microcon (for fraction molecular weight 30000)(manufactured by Millipore) to exchange the buffer. This procedure wasrepeated two times. 20 μl of 100 mM mercaptoethylamine (manufactured byNakaraitesk) was added to 200 μl of anti-human alpha-1-anititrypsin Fab′antibody, the resultant mixture was stirred, and was incubated at 37° C.for 30 minutes. The mixture was concentrated again to 20 μl withMicrocon (for fraction molecular weight 30000) (manufactured byMillipore) and ultrafiltered with 200′ 1 of a 0.1M phosphate buffer with5 mM EDTA (pH 7.5).

25 nmol of tetramethylrhodamine-5-iodoacetamide (manufactured byMolecular Probes) was dissolved in 5 μl of N,N-dimethylformamide(manufactured by Sigma), 75 μl of a 0.1M phosphate buffer 5 mM (withEDTA (pH 7.5)), 5 μl of 1 mM mercaptoethylamine (manufactured byNakaraitesk) and incubated at 37° C. for 10 minutes. This was mixed withmercaptoehylamine-treated anti-human alpha-1-anititrypsin Fab′ antibodyto react in the dark place overnight. The reaction product was subjectedto Sephadex G-25 (manufactured by Amersham Pharmacia Biotech Inc.) toseparate an unreacted fluorescent dye and fluorescently labeledanti-human alpha-1-anititrypsin Fab′ antibody (referred to as onemolecule fluorescently labeled anti-human alpha-1-anititrypsin Fab′antibody in some cases because the antibody is labeled with one moleculeof a fluorescent dye) which were used in the following experiment. Theconcentration of one molecule fluorescently labeled anti-humanalpha-1-anititrypsin Fab′ antibody was determined by absorbancemeasurement using molecular extinction coefficient at 543 nm of 87,000.

(7) Assessment by Isoelectric Focusing with Fluorescent Detection

The resultant one molecule fluorescently labeled anti-humanalpha-1-anititrypsin Fab′ antibody was separated and detected using acapillary electrophoretic apparatus P/ACE5510 manufactured by Beckman.As a capillary, a fused silica capillary (manufactured by GL Science),having an inner diameter of 0.05 mm, an external diameter of 0.375 mm,and a full length of 27 cm, that was covered with polyacrylamidecovalently-bonded to the inner wall was used. Fluorescent detection wasperformed by laser excitation at a position of 20 cm from the anode.After the capillary was filled with an amphoteric carrier solution ofPharmalyte 3-10 (manufactured by Pharmacia BioTec, 40-fold diluted),hydroxypropyl methylcellulose (manufactured by Sigma, hereinafterreferred to as HPMC, final concentration 0.125%), andN,N,N′,N′-tetramethyl ethylenediamine (TEMED, manufactured by PharmaciaBioTec, final concentration 0.6%), an amphoteric carrier solutioncontaining 2×10⁻⁸ M one molecule fluorescently labeled anti-humanalpha-1-anititrypsin Fab′ antibody was injected for 30 seconds throughthe anode at the high pressure mode.

After voltage of 13.5 KV (500 V/cm) was applied for 10 minutes using a20 mM phosphoric acid containing HPMC (final concentration 0.1%) as ananode solution and 20 mM NaOH as a cathode solution, the anode solutionwas injected in the anode side at the low pressure mode whilemaintaining the same voltage, to detect one molecule fluorescentlylabeled anti-human alpha-1-anititrypsin Fab′ antibody which was focusedin the pH gradient carrier. The excitation of the fluorescent dye wasperformed using an argon laser (manufactured by Beckman, Laser Module488) having a wavelength of 488 nm by mounting a 488 nm notch filter(manufactured by Beckman) and a band filter for rhodamine (manufacturedby Asahibunko, especially ordered one) on the filter housing unit. Theobtained results are shown in FIG. 3. As seen from FIG. 3, anisoelectric point of one molecule fluorescently labeled anti-humanalpha-1-anititrypsin Fab′ antibody produced by Escherichia coli wasununiform.

(8) Correction of an Isoelectric Point by a Site-Specific Mutagenesis

In order to correct an ununiformity of the isoelectric point of theanti-human alpha-1-antitrypsin Fab′ antibody, the following DNA primerswhich site-specifically mutates the CH1 region were designed, and amodification of the antibody was performed. In the following experiment,for example, when converting (or having converted) N (asparagine) at the162nd position in the H chain according to the Kabat numbering systeminto D (aspartic acid), the description “H-N162D” is used in some cases.Therefore, the Fab′ antibody in which N (asparagine) at 162nd positionin the H chain according to the Kabat numbering system was convertedinto D (aspartic acid) is expressed as “H-N162D modified Fab′ antibody”and a gene expressing this antibody is expressed as “a gene expressingH-N162D modified Fab′ antibody.”

As a DNA primer, the F5-1 primer (SEQ ID NO: 5 in the Sequence Listing)and the H-N162D-BamHI primer (SEQ ID NO: 6 in the Sequence Listing) wereused. In the following sequences, 5, and 3′ mean a 5′ side and a 3, sideof the primer, respectively, S indicates C or G, M indicates A or C, Rindicates A or G, and W indicates A or T.

F5-1 primer (SEQ ID NO: 5): 5′ SAGGTSMARCTGCAGSAGTCWGG 3′ H-N162D-BamHIprimer (SEQ ID NO: 6) 5′ GCTGGACAGGGATCCAGAGTCCCAGGTCACTGT 3′

A gene fragment in which N (asparagine) at the 162nd position in the Hchain according to the Kabat numbering system was converted into D(aspartic acid) was prepared by performing PCR using these primers andusing the gene expressing anti-human alpha-1-anititrypsin Fab′ antibodyobtained in (2) as a template and the resultant product was digestedwith BamHI. To this digestion product was ligated to a gene fragmentobtained from a low melting point agarose electrophoresis of aBamHI-digested product of the gene expressing an anti-humanalpha-1-anititrypsin Fab′ antibody.

In order to ligate to the same pCANTAB5E plasmid vector as that used in(2), the ligation product was subjected to PCR using the F5-2 primer andthe K3-2 primer having the following sequences, then digested using SfiIand NotI restriction enzymes, and ligated to the pCANTAB5E plasmidvector to make an expression vector containing the gene expressingH-N162D modified Fab′ antibody.

F5-2 primer (SEQ ID NO: 7):5′ CATGTGAACTGACTGGGCCCAGCCGGCCATGGCCGAGGTCCAGCTG CAGCAGTCAGG 3′ K3-2primer (SEQ ID NO: 8) 5′ CCACGATTCTGCGGCCGCACACTCATTCCTGTTGAAGCTCTTTGTAAT 3′

The aforementioned expression vector was transformed into Escherichiacoli HB2151. An antibody was induced as described above, and screeningby ELISA was performed. A plasmid was extracted from the bacteriashowing a positive reaction and transformed into Escherichia coli XL10.The plasmid was extracted from the transformed XL10 bacteria to theamount for use in a sequencing reaction and the base sequence of geneexpressing H-N162D modified Fab′ antibody was confirmed. In preparationof the modified gene, the Pyrobest polymerase having the high fidelity(manufactured by Takara Shuzo) was used as a polymerase. Afterconfirmation of the base sequence, the culturing scale of Escherichiacoli transformed with the expression vector was extended to produceH-N162D modified Fab′ antibody. The antibody obtained was purified by anaffinity column to obtain a purified H-N162D modified Fab′ antibody.

The sequence of the CH1 region and a part adjacent to the C-terminal ofthe CH1 region of H-N162D modified Fab′ antibody which was confirmed bythe aforementioned method is shown below (SEQ ID NO: 9 in the SequenceListing):

  AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWDSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR DCGCSR

In this sequence, the sequence of C-terminal side (right side),VPRDCGCSR, is the amino acid sequence comprising a cysteine residue (C)which is not involved in binding with the L chain, and which wasintroduced into a part adjacent to the C-terminal of the CH1 region.Here, the cysteine residue which is not involved in binding with an Lchain is the one which exists in the C-terminal side of the sequence ofVPRDCGCSR. In addition, a part other than the sequence of VPRDCGCSR isthe sequence of the CH1 region.

The sequence of the corresponding part in the Fab′ antibody beforeH-N162D modification is performed is as follows (SEQ ID NO: 10):

  AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR DCGCSR

When the above two sequences are compared, it is found that theasparagine residue (underlined N) at the 162nd position in the H chainaccording to the Kabat numbering system is converted into aspartic acid(underlined D) after modification. In addition, in this Example, thesequence of a part adjacent to the C-terminal of the CH1 region inH-N162D modified Fab′ antibody (VPRDCGCSR) is the same as the sequenceof a part adjacent to the C-terminal of the CH1 region in an Fab′antibody before H-N162D modification (VPRDCGCSR).

(9) Separation and Detection of a Fluorescently Labeled Fab′ AntibodyHaving a Uniform Isoelectric Point by Fluorescent Detection CapillaryIsoelectric Focusing

The H-N162D modified Fab′ antibody obtained in (8) was fluorescentlylabeled with tetramethylrhodamine-5-iodoacetamide (manufactured byMolecular Probes) according to the same manner as that described in (6),to obtain one molecule fluorescently labeled Fab′ antibody. This onemolecule fluorescently labeled Fab′ antibody was separated and detectedusing the capillary electrophoretic apparatus P/ACE5510 manufactured byBeckman according to the same manner as that of (7). The results thereofare shown in FIG. 4.

As seen from FIG. 4, in the case where one molecule fluorescentlylabeled H-N162D modified Fab′ antibody is subjected to capillaryelectrophoresis, one large peak appeared near an migration time of 29minutes and no substantial peak appeared at other areas. This means thatthe isoelectric point of H-N162D modified Fab′ antibody is uniform.

(10) Modification by Adding an Amino Acid Sequence Comprising a ChargedAmino Acid Residue

In order to add an amino acid sequence comprising a charged amino acidresidue adjacent to the C-terminal of the L chain of the H-N162Dmodified Fab′ antibody obtained in (8), a DNA primer for introducing anamino acid sequence comprising a charged amino acid residue wasdesigned. A gene expressing the H-N162D modified Fab′ antibody which ismodified by adding an amino acid sequence comprising a charged aminoacid residue adjacent to the C-terminal of the L chain (Hereinafter,this gene may be called a gene expressing a charge modified H-N162Dmodified Fab′ antibody.) was obtained by performing PCR using the geneexpressing the H-N162D modified Fab′ antibody as a template and usingthe above-described DNA primers. As a DNA primer, F5-1 primer (SEQ IDNO: 5) described above, F5-2 primer (SEQ ID NO: 7) described above, andK3+5RPS primer (SEQ ID NO: 11) shown below were used.

K3+5RPS primer (SEQ ID NO: 11):5′ GGTGATCGGCCCCCGAGGCCGGTCTACTTGGTCGACTTGGTCGACTAGGTCTAGAAGGACGTGAACACTCATTCCTGTTGAAGCTC 3′

After the gene expressing a charge modified H-N162D modified Fab′antibody is digested with a restriction enzyme of SfiI, or SfiI/NotI,the resultant gene was ligated to a plasmid vector for expressing aprotein (pCANTAB5E plasmid vector or the like). Then, an expressionvector containing the gene expressing a charge modified H-N162D modifiedFab′ antibody was produced.

The aforementioned expression vector was transformed into Escherichiacoli HB2151. An antibody was induced as described above, and screeningby ELISA was performed. A plasmid was extracted from the bacteriashowing a positive reaction and transformed into Escherichia coli XL10.The plasmid was extracted from the transformed XL10 bacteria to theamount for use in a sequencing reaction and the base sequence of thegene expressing a charge modified H-N162D modified Fab′ antibody wasconfirmed. In preparation of the modified gene, the Pyrobest polymerasehaving the high fidelity (manufactured by Takara Shuzo) was used as apolymerase. After confirmation of the base sequence, the culturing scaleof Escherichia coli transformed with the expression vector was extendedto produce H-N162D modified Fab′ antibody which is modified by adding anamino acid sequence comprising a charged amino acid residue(Hereinafter, this may be called a charge modified H-N162D modified Fab′andibody). Then, this antibody was purified by an affinity column toobtain the purified antibody.

The sequence (SEQ ID NO: 12 in the Sequence Listing) of the CL regionand a part adjacent to the C-terminal of the CL region (the C-terminalof the L chain) of the charge modified H-N162D modified Fab′ andibodywhich was confirmed by the aforementioned method is shown below:

  ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPITKSFNRNECSRPSRPSRPSRPSRP

In this sequence, the sequence of the C-terminal side (right side)SRPSRPSRPSRPSRP is the amino acid sequence comprising a charged aminoacid residue which is added adjacent to the C-terminal of the CL region(the C-terminal of the L chain). In this sequence, the sequence of SRPis repeated 5 times, and R (arginine) is the charged amino acid residue.In addition, a part other than the sequence of SRPSRPSRPSRPSRP is asfollows:

  ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPITKS FNRNEC

This sequence is the sequence (SEQ ID NO: 13) of the CL region.

(11) Separation and Detection by Fluorescently Detecting CapillaryIsoelectric Focusing of Immune Complex

The H-N162D modified Fab′ antibody obtained in (8) was fluorescentlylabeled with tetramethylrhodamine-5-iodoacetamide (manufactured byMolecular Probes) according to the same manner as that described in (6).The resultant antibody may be called one molecule fluorescently labeledFab′ antibody having a uniform isoelectric point hereinafter. The chargemodified H-N162D modified Fab′ antibody obtained in (10) was alsofluorescently labeled with tetramethylrhodamine-5-iodoacetamide(manufactured by Molecular Probes) according to the same manner as thatdescribed in (6). The resultant antibody may be called one moleculefluorescently labeled charge modified Fab′ antibody having a uniformisoelectric point.

The concentration of the fluorescently labeled Fab′ antibody having auniform isoelectric point and the fluorescently labeled charge modifiedFab′ antibody having a uniform isoelectric point was determined byabsorbance measurement using molecular extinction coefficient at 543 nmof 87,000. These antibodies were (1) concentrated by centrifugationusing microcon-10 (fractionation molecular weight 10,000) (MilliporeCo.), (2) charged with high pressure steam-sterilized MilliQ water, and(3) centrifuged. Above steps (1)–(3) were repeated twice fordeionization. The resultant product was charged with high pressuresteam-sterilized MilliQ water to reach the final concentration of 80 nM.

The human alpha-1-antitrypsin (Carbiochem Co.) dissolved in highpressure steam-sterilized MilliQ water was (1) concentrated bycentrifugation using microcon-10 (fractionation molecular weight 10,000)(Millipore Co.), (2) charged with high pressure steam-sterilized MilliQwater, and (3) centrifuged. Above steps (1)–(3) were repeated twice fordeionization. The resultant product was charged with high pressuresteam-sterilized MilliQ water to reach the final concentration of 8 μM.

The same amount of the solution of fluorescently labeled Fab′ antibodyhaving a uniform isoelectric point and the solution of the humanalpha-1-antitrypsin were mixed. The mixed solution was mixed with thesame amount of Phrmalyte 3-10 (Amersham pharmacia biotech Co., 20-folddiluted solution) and hydroxypropyl methylcellulose (Sigma Co., finalconcentration: 0.8%). The mixture was kept dark at room temperature for10 minutes to obtain an immune complex. The same procedure was appliedto the fluorescently labeled charge modified Fab′ antibody having auniform isoelectric point to obtain an immune complex. The resultantsolution was separated and detected using a capillary electrophoreticapparatus P/ACE5510 manufactured by Beckman.

The result of the example using the fluorescently labeled Fab′ antibodyhaving a uniform isoelectric point was shown in FIG. 5, while the resultof the example using the fluorescently labeled charge modified Fab′antibody having a uniform isoelectric point is shown in FIG. 6. In FIG.5, a large peak appeared at a migration time of about 22 minutesoverlaps peaks appeared at a migration time of 20–25 minutes. Therefore,separation is insufficient. On the other hand, in FIG. 6, a large peakappeared at a migration time of about 22 minute is clearly separatedfrom peaks appeared at a migration time of 24 minutes and greater.

A schematic view of the method for quantitatively detecting an antigenaccording to the present invention is shown in FIGS. 7A–D, which includesteps for producing the Fab′ antibody having a uniform isoelectricpoint. FIG. 7A shows Escherichia coli in which the gene expressing anFab′ antibody having a uniform isoelectric point of the presentinvention is incorporated and an antibody is induced by acting IPTG(isopropyl-β-D-thiogalactopyranoside) on this Escherichia coli. FIG. 7Bshows the Fab′ antibody having a uniform isoelectric point of thepresent invention produced by this antibody induction. This Fab′antibody having a uniform isoelectric point is purified with an affinitycolumn or the like and, thereafter, fluorescently labeled. FIG. 7C showsthis fluorescently labeled Fab′ antibody having a uniform isoelectricpoint. Upon performing electrophoresis of the immune complex formed bythe fluorescently labeled Fab′ antibody having a uniform isoelectric andan antigen, the data about the relation between migration time andfluorescence intensity, as shown in FIG. 7D, is obtained.

A schematic view of the method for quantitatively detecting an antigenaccording to the conventional method disclosed in JP-A 8-506182 is shownFIGS. 8A–G, which include steps for producing the Fab′ antibody having auniform isoelectric point. By comparing FIGS. 7A–D and FIGS. 8A–G, it isapparent that the conventional method includes a lot of steps, which arecumbersome, to obtain the Fab′ antibody having a uniform isoelectricpoint. Further, in the conventional method, when the isoelectric pointof an antigen is close to that of the fluorescently labeled Fab′antibody having a uniform isoelectric point, migration time of theimmune complex becomes almost the same as those of the excessive antigenand/or antibody. Therefore, peaks are overlapped and detection can notbe performed with high accuracy.

On the other hand, the method for quantitatively detecting an antigenaccording to the present invention, as shown in FIGS. 7A–D, includessimple steps to obtain the Fab′ anibody having a uniform isoelectricpoint. And the method according to the present invention enables adetection with high accuracy even when the isoelectric point of theantigen is close to that of the Fab′ antibody having a uniformisoelectric point.

INDUSTRIAL APPLICABILITY

As explained above, according to the present invention, there can beprovided a method for quantitatively detecting an antigen which enablesa detection with high accuracy even when an isoelectric point of anantigen as an analyte is close to an isoelectric point of afluorescently labeled antibody.

1. A method for quantitatively detecting an antigen in an analyticalsample, said analytical sample suspected of containing an amount ofantigen, which comprises: a) providing a plurality of identical modifiedrecombinant Fab′ antibodies having a uniform isoelectric point, eachsaid modified antibody being specific for the antigen and capable offorming an immune complex with any of the antigen in the analyticalsample, and each said modified Fab′ antibody being recombinantlymodified from a first antigen specific antibody in having: i) at leastone additional charged amino acid residue adjoined to a L-chainC-terminus; ii) a site specific mutation in encoding nucleic acidaltering at least one Fd-chain CH1 region amide side chain-containingamino acid residue in said first antibody to a non-amide sidechain-containing amino acid residue, except cysteine, in said modifiedantibody; iii) a single cysteine residue, which is not involved inbinding of the Fd-chain to the L-chain, in an amino acid sequenceadjoining the Fd-chain CH1 region C-terminus in the modified Fab′antibody; and iv) a fluorescent dye label bound to the singlenon-L-chain binding cysteine residue; b) contacting the plurality ofmodified Fab′ antibodies having a uniform isoelectric point with theanalytical sample in a mixture under conditions sufficient for formationof said immune complexes; c) separating any formed immune complexes fromunbound antibodies and antigen by performing electrophoresis of themixture in a carrier; d) irradiating the electrophoresed mixture in thecarrier with an excitation light which excites the fluorescent dyelabel; e) detecting a level of fluorescence of the separated andirradiated immune complexes or the separated and irradiated unboundantibodies; and f) correlating the detected level of fluorescence withthe amount of antigen in the analytical sample.
 2. The method of claim 1wherein correlating the detected level of fluorescence involvescomparing the detected level of fluorescence with a standard curverelating fluorescence intensity with amount of antigen.
 3. The method ofclaim 1 wherein the electrophoresis is performed by isoelectricfocusing.
 4. The method of claim 1 wherein the electrophoresis isperformed by capillary electrophoresis.